Project Summary/Abstract Inflammatory bowel disease (IBD) is an autoimmune disease that affects the digestive tract. It is strongly correlated with imbalances in members of the gut microbial community. It is not known if the absence or presence of specific microbes contributes to the progression of IBD. Monocultures of gut microbes strongly associated with health or IBD were screened for their ability to produce molecules that are recognized by the immune system to promote inflammation (by inducing TNF?) or to suppress inflammation (by inducing IL10). From this screen, Ruminococcus gnavus was found to produce a potent anti-inflammatory molecule. This work will identify and characterize this anti-inflammatory molecule, determine how it is made, how it is recognized by the immune system, and whether it is present in clinical samples. First, the anti-inflammatory molecule from R. gnavus will be purified from scale-up cultures with activity-guided fractionation. Once pure, the structure will be determined using a combination of NMR, MS and X-ray diffraction. To determine the distribution of the anti-inflammatory molecule and potential analogs among other gut microbes the biosynthetic genes will be determined through gene deletion in R. gnavus, or heterologous expression of genes of interest. Alternatively, strains of R. gnavus (or closely related species) will be screened for those that do not produce the anti-inflammatory molecule, and their genomes will be compared to identify genes associated with anti-inflammatory activity. The anti-inflammatory activity of the molecule is abolished when Myd88 is knocked out; therefore, it is likely recognized by the immune system through a TLR. Each TLR will be knocked out to determine how the anti- inflammatory molecule is recognized by the immune system. The response of the immune system will also be characterized by RNA-seq. The clinical relevance of the anti-inflammatory molecule will be established by searching for its presence in previously collected metabolomic data from patient stool samples. Alternatively, presence and expression of the biosynthetic genes will be determined by mining previously collected metagenomic and transcriptomic data from these same patient samples. Finally, a mouse IBD model will be used to determine the effects of R. gnavus and its purified immunomodulator on disease initiation, severity, and progression. This work will expand our knowledge of how a member of the gut microbiome modulates the immune system and its relevance for IBD, and may also identify potential avenues for therapeutic intervention.